长链非编码RNA GAS5靶向调控miR-424表达抑制宫颈癌HeLa细胞增殖

Long non-coding RNA GAS5 targets the expression of miR-424 and inhibits the proliferation of cervical cancer HeLa cells

摘要:

目的探讨长链非编码RNA GAS5(lncRNA GAS5)调控miR-424抑制宫颈癌细胞增殖的机制。方法预测筛选靶向GAS5的miRNA,用双荧光素酶法检测其荧光素酶活性。分析宫颈癌组织和正常宫颈组织GAS5和miR-424 mRNA水平,及其与患者临床病理特征的关系。检测Ect1/E6E7细胞和HeLa细胞GAS5和miR-424 mRNA水平。将miR-NC、GAS5 RNAi和pCDNA3.0-HA-GAS5质粒转染到HeLa细胞,MTT法及流式细胞术检测细胞增殖和凋亡情况,Western blotting法检测细胞Dnmts、EZH2和Akt3蛋白表达水平。 结果miR-424-mimic可明显降低GAS5-WT荧光素酶活性(P＜0.05),表明GAS5与miR-424存在靶向调节作用。宫颈癌组织GAS5、miR-424 mRNA表达低于正常宫颈组织(P＜0.05)；是否转移、不同FIGO分型患者间GAS5和miR-424表达存在差异(P＜0.05)。HeLa细胞GAS5和miR-424 mRNA表达低于Ect1/E6E7细胞(P＜0.05)。与阴性对照组比较,HeLa细胞GAS5、miR-424 mRNA、细胞凋亡率、Dnmts和EZH2表达GAS5 RNAi组降低,GAS5质粒组增加；细胞增殖率和Akt3表达GAS5 RNAi组增加,GAS5质粒组降低(P＜0.05)。 结论lncRNA GAS5通过靶向调控miR-424的表达抑制宫颈癌HeLa细胞增殖。

Abstract:

To investigate the mechanism of Long Non-coding RNA GAS5 regulating MiR-424 to promote the proliferation of cervical cancer cells. MethodsThe miRNA targeting GAS5 was predicted and screened. The luciferase activity was detected by the dual luciferase method. The mRNA levels of GAS5 and miR-424 in cervical cancer tissue and normal cervical tissue and their relationship with clinicopathological characteristics of patients were analyzed. The mRNA levels of GAS5 and miR-424 were detected in Ect1/E6E7 cells and HeLa cells. The miR-NC, GAS5 RNAi and pCDNA3.0-HA-GAS5 plasmids were transfected into HeLa cells, and the cell proliferation and apoptosis were detected by MTT and flow cytometry, and the Dnmts, EZH2 and Akt3 proteins were detected by Western blotting. ResultsmiR-424 could significantly reduce the luciferase activity of GAS5-WT (P＜0.05), indicating that GAS5 and miR-424 have a targeted regulatory effect. The mRNA expressions of GAS5 and miR-424 in cervical cancer tissues were lower than those in normal cervical tissues (P＜0.05). The low expression rates of GAS5 and miR-424 in cervical cancer patients with metastasis or FIGO type Ⅱb-Ⅲa were higher than those in patients without metastasis, type Ⅰb-Ⅱa patients. The mRNA expressions of GAS5 and miR-424 in HeLa cells were lower than those in Ect1/E6E7 cells (P＜0.05). Compared with the negative control group, GAS5, miR-424 mRNA, apoptosis rate, Dnmts and EZH2 expression in HeLa cells decreased in GAS5 RNAi group, but increased in GAS5 plasmid group; cell proliferation rate and Akt3 expression in GAS5 RNAi group increased, but decreased in GAS5 plasmid group (P＜0.05). ConclusionLong non-coding RNA GAS5 inhibits the proliferation of cervical cancer HeLa cells by targeting the expression of miR-424.